Tryptophan hydroxylase from rat mesencephalic tegmentum has been purified by sequential chromatography on Blue-Sepharose, DEAE, and calmodulin-Sepharose. The hydroxylase is excluded from Blue-Sepharose and is eluted from DEAE with a step-wise NaCl gradient. Finally, tryptophan hydroxylase binds to calmodulin-sepha-rose and is eluted with EGTA. The purification scheme is rapid and yields an enzyme with a specific activity of 3.5 nmol 5HTP/mg min, representing a 61-fold purification with 8% recovery. It appears that tryptophan hydroxylase adsorbs to calmodulin-Sepharose through hydrophobic interactions. Tryptophan hydroxylase also binds to the hydrophobic matrix phenyl-Sepharose and is eluted with ethylene glycol.